Area of Interest

Our work is focused on developing new tools for analyzing biological macro­molecules,
including DNA and proteins. Specifically, we are interested in fabricating miniaturized
systems for mutation analysis (diagnostics), isolating cells from mixed populations,
developing systems for high throughput protein analyses and evolving new technologies
for DNA sequencing as part of the Human Genome Initiative. In order to build devices
specifically for molecular analyses, our research spans many sub-areas, such as polymer-based
micro- and nano­machining, fluorescent probe development, construction of ultra­sensitive
detection apparati and nano-biology (performing molecular biological reactions in
small volumes). In addition, we are currently working with collaborators in several
areas, such as mechanical engineering, molecular biology, surface science, materials,
organic chemistry and mass spectrometry. Provided below is a short description of
a few of our many projects. To facilitate these multi-discplinary efforts, our group
is part of the Center for BioModular Multi-Scale Systems on the campus of LSU, which
is focused on building enabling tools for discovery in the life sciences and clinical
applications in medicine.

Mutations (wrong order of the DNA building blocks) in coding regions of genes can serve
as important markers for the early diagnosis of genetically related diseases, such
as breast cancer, cystic fibrosis and sickle cell anemia. One of our projects involves
developing novel tools for the efficient detection of mutations in K-ras genes associated with colorectal cancer. In addition, we have a project focused on
capturing cancer cells in circulating blood which carry cell surface markers associated
with breast cancer. These projects (funded by the National Cancer Institute) involve
collab­or­ations with our Center forAdvanced Micro­structures and Devices (CAMD) to fabricate high-aspect-ratio molding tools to hot-emboss devices in various polymers,
such as poly (methyl­methacrylate) or polycarbonate. These devices are used to prepare
the DNA sample (isolate DNA from various body fluids), amplify the number of DNA molecules
to be analyzed via PCR, discriminate the normal DNAs from mutant DNAs using mutation
screening reactions, sort the DNA by size in an electric field and detection using
miniaturized laser-induced fluorescence fiber optic detectors of DNA tagged with colored
dyes possessing unique photophysical properties. Figure 1 shows some of the devices we have fabricated using our established micro- and nano-manufacturing
techniques.

Another example of our research is focused on developing tools for the capture of low
abundant cells from mixed populations in clinical samples. For example, devices are
being constructed to collect rare circulating tumor cells from whole blood using microfluidics.
Circulating tumor cells can be present at a level of 1-10 cells per milliliter of
blood with the level of normal cells (red blood cells and white blood cells) being
substantially higher (10 million per milliliter of blood). Therefore, we have developed
a device to capture these rare cells using an affinity capture method that can pre-select
the tumor cells with high efficiency and clear the red and white blood cells. The
tumor cells are then quantitatively counted using a non-labeling process, which consists
of running the cells through a pair of electrodes and measuring the solution conductance.Figure 2 details the system and shows micrographs of tumor cells captured and counted from
whole blood using this system.

Our research is also directed toward improving the process of identifying proteins
comprising the proteome. We are developng an integrated system that can pre-select
a certain sub-population of proteins from the proteome and then efficiently identify
those proteins using mass spectrometry interfaced to a fluidic system. The system
consists of the following processing steps: (1) Solid-phase capture using an affinity
bed of the protein sub-population to be analyzed; (2) two-dimensional electrophoresis
platform for isolation of the constituent proteins; (3)solid-phase bioreactors to
digest the isolated proteins into peptide fragments; (4) capillary electrochromatography
unit for the high-resolution separation of the generated peptides and (5) interface
to a mass spectrometer for identifying the peptides and consequently, the proteins.
An example of our system and the results generated for protein fingerprinting is shown
in Figure 3.

Another interesting project involves developing single molecule detection methods for
the real time molecular screening of mutated DNAs. We have developed an assay that
can provide molecular signatures of disease states in < 5 min using single molecule
detection. The assays utilize a ligation reaction, which forms a molecular beacon
that contains two fluorescent dyes at their ends. Using a microfluidic device and
confocal fluorescence detection, we can obtain information on the presence of point
mutations in genomic DNA in less than 5 minutes, nearly 20 times faster than what
can be done with conventional instrumentation. In Figure 4 is shown data accumulated using this assay format.

Awards & Honors

William L. and Patricia Senn, Jr. Professor

Director, Center for BioModular Multi-Scale Systems

A. Benedetti-Pichler Award in Microchemistry

Charles E. Coates Award for Outstanding Contributions to Chemical/Engineering Research
in Louisiana, 2001